Dulbecco’s modified eagle medium (DMEM, PYG0073, Boster), Roswell park memorial institute 1640 medium (RPMI-1640, PYG0006, Boster), GenOpti medium (CT007.500, M&C gene technology ltd.), Fetal Bovine Serum (C04001-500, VivaCell), PBS (03.15018C, EallBio), M-CSF Protein, Mouse (HY-P7058, MedChemExpress), Polyinosinic-polycytidylic acid (Poly(I:C), HY-107202, MedChemExpress), Trizol (111003, Alcatel), VAMNE Magnetic Universal Total RNA Kit (ROA3301-01) Protease inhibitor cocktail (C0001, TargetMol), StarFect transfection reagent (C101-10, GenStar), StarScript III All-in-one RT (A230-10, GenStar), 2×RealStar Fast SYBR qPCR Mix (Low ROX) (A304-10, GenStar), Sodium chloride (C090701, YangGuangBio), IRF3 (phospho Ser396) antibody (GTX86691, Human/Mouse, 1:1000, GenTex), Anti-IRF3 (phospho S386) antibody (ab76493, Human 1:1000, Abcam), Anti-IRF3 antibody (ab68481, Human/Mouse 1:1000, Abcam), Hepatitis B Virus Core Antigen (ab8639, 1:100, Abcam), Hsp90 (5D6) Mouse mAb (251211, Human/Mouse, 1:1000, Zenbio), TMEM173/STING Polyclonal antibody (19851-1-AP, Human/Mouse, 1:2500, Proteintech), Universal Antibody Diluent (02.13451-100, EallBio) FITC anti-mouse CD3 (100204, Mouse, 1:200, BioLegend), PE anti-mouse CD4 (100408, Mouse,1:600, BioLegend), PerCP/Cyanine5.5 anti-mouse CD8a (100734, Mouse, 1:150, BioLegend), APC anti-mouse IFN-γ (505810, Mouse, 1:100, BioLegend), Zombie NIR™ Fixable Viability Kit (423105, 1:5000, BioLegend), 4% Paraformaldehyde Fix Solution (G1101-500ML, Servicebio), 2′3′-cGAMP ELISA Kit (501700, Cayman Chemical), LumiKine™ Xpress mIFN-β 2.0 (luex-mifnbv2, Mouse, InvivoGen), LumiKine™ Xpress hIFN-β 2.0 (luex-hifnbv2, Human, InvivoGen), Nuclear and Cytoplasmic Protein Extraction Kit (P0028, Beyotime). Human hepatitis B virus e antigen (HBeAg) ELISA Kit (ZC01803, Wantai biopharm), Human hepatitisB surface antigen (HBsAg) ELISA Kit (ZCY0503, Wantai biopharm), Quantitative PCR Diagnostic Kit for HBV-DNA (PCR-Fluorescence Probing in One-Tube) (B001, NaGene Diagnosis), ISD was synthesized as previously described by heating equimolar amounts of sense DNA and anti-DNA oligo acids to 95 °C for 5 min and then cooling to room temperature. The Sophora tonkinensis are derived from the dried roots and rhizomes of the legume Sophora tonkinensis Gagnep., pAAV/HBV1.2 plasmid was from XJ Wang (Academy of Military Medical Sciences).

The botanical drug Sophorae Tonkinensis Radix et Rhizoma used in this study consists of the dried roots and rhizomes of Sophora tonkinensis Gagnep. (Fabaceae; batch no. 23011701, Taizhou Baicao Chinese Medicine Drinks Slice Co., Ltd.). The botanical material was excavated in Guangxi, China, in September 2022 and is deposited in the Department of Liver Diseases, Chinese PLA General Hospital, where it was authenticated as authentic by Professor Xiao-He Xiao. The crude drug appears as irregular, nearly circular slices 2–4 mm thick, with an outer surface brown to dark brown; the cortex is light brown and the xylem pale yellow in cross-section. It emits a characteristic bean-like odor and tastes extremely bitter (Heinrich et al., 2022).

For qualitative analysis of the active constituents in STE, reference standards of oxymatrine, oxysophocarpine, matrine, trifolirhizin, and maackiain were dissolved in methanol to prepare individual stock solutions. Weigh 2 g of STE precisely into a volumetric flask, add 20 mL of water, and prepare the test sample solution by sonicating for 60 min. Freeze-dry the solution. The test sample solution was prepared by accurately weighing 50 mg of lyophilized powder into a 10-mL volumetric flask, adding 8 mL of water, and sonicating for 30 min. After cooling to room temperature, the mixture was diluted to volume with water, mixed thoroughly, and filtered; the filtrate was used for analysis. Chromatographic separation was performed on a PFchrom EP C18 column (4.6 × 250 mm, 5 μm; FX-063) maintained at 35  °C. A gradient elution program was employed with acetonitrile and 4 mmol/L dipotassium hydrogen phosphate as mobile phases: 8%–10% acetonitrile (0–4 min), 10%–15% (4–15 min), 15%–27% (15–18 min), 27%–37% (18–30 min), 37%–75% (30–32 min), held at 75% (32–40 min), returned to 8% (40–41 min), and held at 8% until the end of the run. The flow rate was 1.0 mL/min, the detection wavelength was set at 215 nm, and the injection volume was 10 μL.

C57BL/6J male mice, weighing 18–20 g, aged 8 weeks, were supplied by Beijing SPF Biotechnology Co., Ltd. and were allowed to acclimatize for 1 week prior to the experiment. The mice were kept in a pathogen-free environment while being given adequate light and darkness as well as water and food. Animal experiments were permitted by the Animal Ethics Committee of the Fifth Medical Center of the General Hospital of the Chinese People’s Liberation Army (Approval Date ID: IACUC-2024-0006).

In vivo experiments, male C57BL/6J mice were constructed as HBV models by hydrodynamic injection of 20 μg pAAV-HBV1.2 plasmid. Different concentrations of STE (0.2 and 0.4 g/kg) or ETV (1 g/kg) were given daily (n = 6 per group), and the control group was injected with saline in the tail vein and given the same concentration of vehicle daily. It was given continuously for 4 weeks, and the serum levels of HBeAg, HBsAg and HBV-DNA in mice were measured weekly. The content levels of HBsAg (ZCY0503, Wantai biopharm) and HBeAg (ZC01803, Wantai biopharm) in the serum of mice were detected by Elisa, and the level of HBV-DNA was detected by PCR-Fluorescence Probing in One-Tube. The mice were executed at week 4, with serum, liver and spleen tissues collected (Sang et al., 2017).

BMDMs were derived from bone marrow of healthy wild-type mice and cultivated in DMEM (including 10% FBS and 1% P/S) with MCSF, and the BMDMs were seeded at a plate density of 1.2*10⁶ cells/mL, and planted in 12-well plates at 1 mL in each well. THP-1 (purchased from Procell, CL-0233) was purchased from Servicebio (BNCC358410) and cultured in 1640 at a seed plate density of 1.2*10⁶ cells/mL, with 0.5 mL per well planted in 24-well plates. HEK-293 (purchased from Procell, CL-0001) was cultured in medium including DMEM at a seed plate density of 5.5*10⁵ cells/mL, with 0.5 mL per well planted in 24-well plates (Wang et al., 2019).

BMDMs or THP-1 were first pretreated with various concentrations of STE or vehicle for 1 h, and then stimulated with ISD (2.5 μg/mL) or Poly(I:C) (2 μg/mL) for 2.5 h. Whole-cell lysates were collected for immunoblotting; the cells were lysed with Trizol at 4 h of stimulation with ISD or Poly(I:C), and the relevant genes were detected for transcription levels; at 8 h of stimulation with ISD or Poly(I:C), cell supernatants were collected to detect IFN-β levels. For plasmid transfection experiments, HEK-293 cells (24-well plate) were transfected with HA-cGAS (0.5 μg), HA-STING (0.5 μg), HA-TBK1 (0.5 μg), or HA-IRF3(0.5 μg) plasmids, respectively, and STE or vehicle was administered 15 h later, and samples were collected 6 h later for and fluorescence quantitative qPCR. The plasmids used in this experiment were purchased from YouBio.

HepG2 2.2.15 cells were seeded at 2.0*10⁵ in Transwell chambers. PMA-induced THP-1 was seeded at 1.2*10⁶ in 24-well plates. After the cells were adhered to the wall, the cells were treated with 1 mg/mL STE, and then co-cultured with the chambers containing HepG2 2.2.15 cells. After 24 h, the cell supernatants were collected, Elisa was used to detect the expression of HBsAg and HBeAg, and THP-1 cells were lysed with Trizol, the transcript level of IFN-β was detected using qPCR (Wu et al., 2024).

BMDMs or THP-1 were seeded at a plate density of 1.2*10⁶ cells/mL, and planted in 96-well plates at the corresponding concentration of 0.1 mL per well and left for 12 h. After 12 h, different concentrations of STE (0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 4 mg/mL, 8 mg/mL, 16 mg/mL, 20 mg/mL) or vehicle were given, and after a further 12 h, 10 μL of CCK-8 was added to all well of the well plates, which were then incubated at 37 °C in an incubator for 50 min. The optical density (OD) values were measured at a wavelength of 450 nm using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc.) to assess cell viability (Ren et al., 2022).

The collected whole-cell lysate was heated at 95 °C for 10 min to denature the proteins, followed by brief centrifugation at 12000 rpm for 5 min to remove any insoluble debris. Protein concentration was measured using the BCA Protein Assay Kit (Solarbio,PC0020) according to the manufacturer’s instructions. The samples were loaded onto a 10% SDS-PAGE gel and proteins were separated according to molecular weight at 85 V for 30 min, then adjusted to 140 V and run for an additional 1 h. Proteins were subsequently transferred to PVDF membranes at 450 mA for 3 h using a transfer buffer composed of 25 mM Tris, 192 mM glycine, and 20% methanol. The PVDF membrane was blocked in 5% skim milk powder in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature. The membrane was then washed three times with TBST for 5 min each, followed by incubation with the corresponding primary antibody diluted in 5% skim milk powder in TBST at 4 °C overnight. After incubation, the membrane was washed again three times with TBST for 5 min each, and then incubated with HRP-conjugated secondary antibody (Cell Signaling Technology) diluted 1:5000 in 5% skim milk powder in TBST for 1 h at room temperature. The membrane was washed three times with TBST for 5 min each, and the proteins were visualized using an ECL detection system (Wang et al., 2020).

The RNA Extraction Kit used was the VAMNE Magnetic Universal Total RNA Kit (Vazyme, ROA3301-01), and the Reverse Transcription Kit was the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Catalog Number: 4368813). The reverse transcription was performed according to the manufacturer’s instructions, using a total of 1 μg of RNA for each sample. The RT-qPCR was performed using the SYBR Green qPCR Master Mix (Low ROX) (A304-10, GenStar) on a StepOnePlus Real-Time PCR System (Applied Biosystems). The qPCR cycling conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 60 °C for 30 s and 72 °C for 1 min. The primers used for the target gene were designed to amplify a specific region of the gene, and their sequences were as show in Supplementary Figure S2. Data analysis was performed using the ΔΔCt method, which involves calculating the relative expression levels of the target gene normalized to the reference gene (β-Actin) and relative to a calibrator sample. ence (Yao et al., 2025).

The levels of IFN-β in cell culture supernatants or mouse serum were determined using the IFN-β ELISA kit (Luex-mifnbv2, InvivoGen). The assay was performed in accordance with the manufacturer’s instructions. Briefly, samples were added to wells of a 96-well plate in triplicate. Following sample addition, the Lucia-Conjugate was introduced to each well. The plate was then incubated at 37 °C for 90 min to allow for the specific binding of IFN-β to the conjugate. After the incubation period, the wells were washed five times with the Washing Buffer provided in the kit to remove any unbound conjugate. Subsequently, the Substrate Solution was added to each well. The luminescence intensity, indicative of the IFN-β concentration, was measured using a luminometer or a multi-mode microplate reader equipped with luminescence detection capabilities. Data were analyzed by comparing the luminescence values of the samples to a standard curve generated using known concentrations of IFN-β. The concentration of IFN-β in the samples was then extrapolated from this standard curve.

The levels of HBsAg and HBeAg were measured following the protocol outlined in the HBeAg ELISA kit instructions, with slight modifications tailored to the detection of HBsAg. Specifically, 5 μL of each test sample, as well as negative and positive controls, were pipetted into the corresponding wells of a microplate. To each well, 50 μL of the enzyme conjugate reagent was added. The plate was then incubated at 37 °C for 30 min to facilitate the binding of the enzyme conjugate to the antigen. After incubation, the wells were washed five times with the washing buffer to remove any unbound enzyme conjugate. Next, 50 μL each of the Chromogen Solution A and Chromogen Solution B were added to the wells. The plate was incubated at 37 °C in the dark for 15 min to allow for color development. Following this, 50 μL of the stop solution was added to each well to terminate the reaction. The absorbance was then measured at a wavelength of 450 nm using a microplate reader to determine the concentration of HBsAg in the samples. The optical density values were used to calculate the antigen levels by comparing them to a standard curve constructed with known concentrations of HBsAg and HBeAg.

The concentration of HBV-DNA was determined using a quantitative PCR-based assay. Initially, 3 μL of nucleic acid release agent was aliquoted into each PCR reaction tube. Subsequently, 3 μL of each test sample, as well as 3 μL of the HBV-negative control, HBV borderline positive control (OT), and HBV quantitative standards (OT) I to IV, were added to their respective PCR tubes. The contents were thoroughly mixed to ensure homogeneity. Following the addition of samples and standards, the samples underwent a lysis procedure to release the nucleic acids. After lysis, 32 μL of PCR mix was added to each tube. The tubes were then centrifuged to ensure all components were at the bottom of the tubes, and were subsequently loaded onto the PCR machine for amplification. The presence and quantity of HBV-DNA were determined by analyzing the fluorescent signal generated during the PCR process using a real-time PCR instrument. The data were analyzed by comparing the cycle threshold (Ct) values of the samples to a standard curve constructed with known concentrations of HBV-DNA.

The ratios of immune cells and interferon in mouse spleen tissues after STE and ETV interventions were examined using flow cytometry. After rinsing the collected spleen tissues with PBS, the tissues were dissociated in RPMI-1640 medium containing 2% FBS (Fetal Bovine Serum). The cell suspension was then filtered through a 200 μm nylon mesh to obtain a single-cell suspension. Subsequently, Zombie NIR™ Fixable Viability Kit 423105, 1:5000, BioLegend) was added to the cell suspension and centrifuged to lyse and remove erythrocytes. The remaining cells were washed with PBS. The cells were fixed with 4% Paraformaldehyde (G1101-500ML, Servicebio) for 10 min at room temperature. After fixation, the cells were washed with PBS and stained with the appropriate antibody for 50 min at 4 °C in the dark. Following incubation, the cells were washed with PBS to remove unbound antibody. Finally, all samples were analyzed by flow cytometry, and the data were processed and analyzed using FlowJo 10.8.1 software (FlowJo LLC).

Statistical analysis was performed by GraphPad 8.0, and all data were showed as Mean ± SD, when p < 0.05, statistical differences were considered to exist. Statistical analyses were performed on multiple samples using one-way ANOVA with Dunnett’s post hoc test.

In this study, we explored the impact of STE on the activation of the cGAS-STING pathway. We initially assessed the effect of STE at varying concentrations (0–20 mg/mL) on the viability of BMDMs and THP-1 cells to establish a safe concentration range. Our findings indicated that STE concentrations up to 2 mg/mL did not affect cell viability, whereas concentrations of 4 mg/mL or higher began to exhibit cytotoxic effects (Supplementary Figure S2A,B). Subsequently, BMDMs were pretreated with STE at concentrations of 0.25, 0.5, and 1 mg/mL for 1 hour, followed by transfection with ISD to activate the cGAS-STING pathway. We then measured the expression levels of p-IRF3, p-STING, STING, and IRF3 using immunoblotting. The results demonstrated that STE promoted the phosphorylation of IRF3 and STING in a concentration-dependent manner (Figure 1A) and enhanced the production of IFN-β induced by ISD (Figure 1B). qPCR analysis further revealed that STE significantly upregulated the expression of IFN-β, CXCL10, IFIT1, ISG15, IL-6, and TNF-α (Figures 1C–H), indicating an enhancement of cGAS-STING activation in BMDMs. Similarly, the activating effect of STE on the cGAS-STING pathway in THP-1 cells was investigated. The results showed that STE promoted the phosphorylation of IRF3 and STING, increased IFN-β production, and elevated the transcription levels of IFN-β, CXCL10, IFIT1, ISG15, IL-6, and TNF-α in THP-1 cells stimulated with ISD (Figures 1I–P). Collectively, our data indicate that STE significantly enhances cGAS-STING activation and the induction of interferon-stimulated genes and inflammatory cytokines in both BMDMs and THP-1 cells.

We next sought to determine whether STE influences the RIG-I pathway, another crucial antiviral signaling mechanism that detects intracellular pathogenic RNA. To investigate the potential regulatory effects of STE on the RIG-I pathway, we established an activation model using Poly(I:C) transfection. BMDMs were treated with STE for 1.5 h, followed by transfection with Poly(I:C) for an additional 2.5 h. Our findings indicated that STE did not alter the levels of p-IRF3, the amount of IFN-β in the supernatant, or the expression of downstream interferon-stimulated genes (Figures 2A–H). Similarly, in THP-1 cells, STE had no impact on Poly(I:C)-induced pathway activation or the upregulation of gene expression (Figures 2I–L). These results suggest that STE does not influence the activation of the RIG-I pathway.

We further explored the mechanism by which STE regulates the cGAS-STING pathway. Our data demonstrated that STE could promote the translocation of IRF3 to the nucleus, confirming its effect on cGAS-STING activation (Figures 3A,B). To investigate the mechanism of STE in promoting cGAS-STING pathway activation, HEK293 cells were transfected with plasmids expressing cGAS, STING, TBK1, or IRF3-5D, which is a constitutively active mutant of IRF3. After administration of STE, the expression of IFN-β was detected by qPCR. The results indicated that STE promoted IFN-β expression induced by cGAS overexpression but had no significant effect on STING, IRF3-5D, and TBK1-induced increases in IFN-β expression (Figures 3C–F). This suggests that STE may act upstream of STING to promote cGAS-STING activation. We next examined the effect of STE on the production of 2′3′-cGAMP, which is synthesized by cGAS upon DNA stimulation. The results showed that 2′3′-cGAMP synthesis could be dose-dependently enhanced by STE in both BMDMs and THP-1 cells (Figure 3G; Supplementary Figure S3A). These findings indicate that STE promotes cGAS-STING pathway activation by facilitating the synthesis of 2′3′-cGAMP mediated by cGAS.

Given the crucial role of the cGAS-STING pathway in controlling infections of multiple viruses, including HBV, we investigated the inhibitory effect of STE on HBV in vitro. We established a co-culture system to verify the anti-HBV activity of STE. Transwell chambers seeded with HepG2 2.2.15 cells were placed on top of 24-well dishes containing THP-1 cells treated with STE or vehicle. After 24 h, the levels of HBeAg, HBsAg, and IFN-β were detected. The results demonstrated that STE significantly increased the expression of IFN-β when added to the THP-1 and HepG2.2.15 co-culture system (Figure 3H). Additionally, STE effectively reduced the levels of HBeAg and HBsAg (Figures 3I,J, S3B). Specifically, the reduction in HBsAg levels was 50%, respectively, compared to the control group. However, STE had no significant effect on HBsAg levels when directly applied to HepG2.2.15 cells, suggesting that STE enhances cGAS-STING activation and IFN production in THP-1 cells, thereby indirectly suppressing HBsAg and HBsAg level in HepG2.2.15 cells.

Next we tested if SET inhibited HBV replication in vivo. The HBV mice model was constructed by hydrodynamic injection of pAAV-HBV1.2 plasmid, the mice were divided into 4 groups: normal saline group, STE (0.2 g/kg) group, STE (0.4 g/kg) group, or ETV (1 g/kg) (Figure 4A). The results found that HBsAg levels in serum of mice decreased after administration of STE, the effect was even better than ETV (Figure 4B). While serum HBeAg and HBV DNA level showed downregulation after administration of STE or ETV, the therapeutic effect of high dose of STE was similar to that of ETV group (Figures 4C,D). Meanwhile, we performed immunohistochemical staining of the collected liver tissues to detect the number of HBcAg-positive hepatocytes, there were fewer positive cells in mice of STE or ETV group than the model group (Figures 4E,F). These results showed that STE can significantly suppress HBV replication in mice. Next, we examined the expression levels of ALT and AST in the serum of mice (Figures 4G,H). The data found that after the administration of STE, the levels of ALT and AST appeared to be reduced compared to the model group, which indicated that hepatic STE could improve the liver histopathology in mice.

We next assessed whether STE promotes the expression of key genes involved in cGAS-STING pathway activation. Gene expression of STING, IFN-β, TNF-α and TRIM12C in liver tissue was significantly induced in mice treated with Ste (Figures 5A–D) Specifically, the expression of STING, IFN-β, TNF-α, and TRIM12C genes was significantly increased in Ste liver tissue in the high-dose group compared with the control group, indicating that Ste enhanced the activation of cGAS-STING signaling in vivo. We also examined the frequency of CD4⁺ and CD8⁺ T cells in the spleen of HBV mice. In our study, we observed a significant increase in the frequency of CD4⁺ T and CD8⁺ T cells in the STE groups compared with the control group (Figures 5E,G). The percentage increase was 35% for CD4⁺ T cells and 40% for CD8⁺ T cells. Additionally, we evaluated the level of IFN-γ in CD4⁺ and CD8⁺ T cells, and the frequency of IFN-γ⁺ CD4⁺ and IFN-γ⁺ CD8⁺ T cells. The frequency of IFN-γ⁺ CD4⁺ and IFN-γ⁺ CD8⁺ T cells in the STE group was increased by 3-fold and 2-fold, respectively, compared to the model group (Figures 5F,H,I). These results suggest that STE enhances the activation of both CD4⁺ and CD8⁺ T cells in HBV mice.

Taken together, these results suggest that STE inhibits HBV replication, improves liver histopathology, and promotes cGAS-STING pathway activation in vivo. These results suggest that the inhibition of HBV by STE may be associated with the promotion of cGAS-STING signaling activation.